These projects take advantage of the laboratory's expertise in studying molecular interactions and molecular structure. For project (1) indicated above, we have developed a strategy for generating disulfide-trapped peptide/MHC-I molecules and have expressed, refolded, purified, and crystallized several of these. In particular, we have examined H2-Dd molecules in which a residue in the binding cleft has been mutated to cysteine and have refolded these with peptides containing cysteine in appropriate positions. X-ray crystallographic structures of these confirm that the disulfide-trapped peptides are stably fixed in the peptide binding groove. Versions of these, produced with truncated peptides, indicate that these molecules are peptide-stabilized in part of the peptide binding groove but are relaxed in the positions of truncation. Binding studies indicate that these molecules should allow mapping of sites of interaction with various cellular and viral chaperones. Using a slightly different approach, we have produced several variations of MHC-I molecules with novel intrachain disulfide bonds, specifically HLA-B*18:01, H2-Dd and H2-Ld. Although we originally expected these to exhibit greater thermal stability, studies underway indicate that the stability is also related to the particular peptide bound by these MHC molecules. Further studies to investigate not only the stability, but the biological activity and utility as tetramer staining reagents are underway. For (2) above, collaborative with the Berzofsky laboratory, we explored unique peptides generated as a result of vaccine immunization of mice in which cathepsin S sites were manipulated to alter cross-presentation. Three peptides (IGPGRAFYVI, IGPGRAFYV, and IGPGRAFYT) from the HIV-MNgp120 protein were identified as presented by the H2-Dd molecule. Our laboratory examined the X-ray structures of these unique H2-Dd/peptide complexes. The three-dimensional structures explain the differential binding of the peptides, and also explain the behavior of their respective tetramers in identifying CD8 T cell populations evoked by vaccination. For (3), carried out collaboratively with Shihoko Komine-Aizawa and Mitsuo Honda, explores the CD8 T cell response to immunization of mice with recombinant BCG-based vaccines that encode the Ag85B protein of M. tuberculosis. Several H2-Kd-restricted Ag85B peptides were identified (YYQSGLSIV and YQSGLSIVM) and we have crystallized and determined the X-ray structures of these. These structures show that variant peptides can bind to MHC molecules like H2-Kd in novel conformations, and offer structural insight into cross-reactive and non-cross-reactive T cell populations primed by the new vaccines. Peptide/H2-Kd tetramers identify two distinct T cell populations elicited by the Ag85B vaccine. In particular, the YQSGLSIVM peptide binds in a non-canonical orientation with its C-terminal residue extending beyond the peptide binding groove. In the fourth sub-project listed above, we have successfully engineered the LAG-3 protein and its fibrinogen like protein 1 ligand as well as various MHC-II ligands. These new reagents will provide the basis for studying the interaction of LAG-3 with different MHC-II conformations and for evaluating the role of its interaction with FGL1.